Antigen specific receptors of B and T lineage cells require the somatic assembly of V, D, and J gene segments during lymphocyte development. Immunoglobulin and T cell receptor loci are targeted by a common V(D)J recombinase in a cell type- and developmental stage-specific manner. The fundamental control of this process occurs at the level of alterations in chromatin structure that confer locus """"""""accessibility"""""""" to the RAG1/2 complex. The immunoglobulin heavy chain locus (Igh) is the first to rearrange during B cell differentiation and accessibility at different stages is limited to discrete domains. The cis-acting elements and signaling pathways that regulate accessibility throughout the lgh locus are yet to be fully elucidated. Since V(D)J recombination is required for normal lymphocyte development, defects in the process result in both subtle and catastrophic immune deficiencies. Thus, understanding the underlying mechanisms has obvious implications to health and disease. Our long term goals are focused on the control of Igh locus accessibility within the nearly 3 megabase region containing approximately 200 Vh gene segments. In this application, we propose to extend our studies of a candidate control region in the 5' boundary of the lgh locus that exhibits early B cell specific chromatin remodeling. This novel region interacts, both in vitro and in vivo, with factors that have been implicated in Igh accessibility and rearrangement. We propose to examine sorted early B cell subsets from bone marrow to determine the precise stage of B cell development at which this element is active. Gel shift and chromatin immunoprecipitation approaches will be used to define the factors that are recruited to the multiple sites mapped within this region. The function of this putative regulatory element will be tested by both genomic deletion and studies of BAC transgenes containing putative control regions and Vh segments of the 5' region of the locus. We will use a novel cell line model as well as primary bone marrow cells to determine the mechanism through which IL-7 receptor signaling selectively mediates D-distal (5') Vh gene accessibility.
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